Lecture theme 2 Structure of chromatin

Question 1

Regulation involves short & long-term processes.

Answer 1

 Short-term
proteins interact transiently with DNA elements, transient changes in chromatin structure
 Long-term
permanent changes in chromatin structure

Question 2

Gene regulation depends on three fundamental elements:

Answer 2

 DNA needs to be assessable to regulating proteins

 DNA has to contain elements that can be influenced by regulating proteins

 DNA and regulatory proteins need to interact

Question 3

ASPECTS THAT INFLUENCE TRANSCRIPTION.

Answer 3

1. Chromatin conformation
    DNA is either tightly or loosely packaged; this determines accessible of DNA control elements to regulatory proteins
    chromatin structure can be affected by: chemical modifications to DNA and/or histones, binding of proteins, binding of small RNA molecules
2. Control elements on DNA
    large variety: basal promoter, enhancers, silencers etc.
    combination of different TFs with different control regions; provide specificity

Transcription factors (TFs) 
 interact with DNA control elements 
 interact with other TFs and co-factors 
 interact with RNA polymerases

Question 4

Chromatin remodeling can involve the following changes:

Answer 4

(a) change in nucleosome structure
(b) displacement of nucleosome along the DNA
(c) complete disassembly of a nucleosome

Question 5

Histone modifications to chromatin.

Answer 5

 acetylation
  methylation
  sumoylation
  ubiquitination
  phosphorylation

Question 6

1. Acetylation of histones.

Answer 6

Lysine, H3 and H4
 catalysed by histone acetyl-transferase (HAT) enzymes
 acetylation reduces positive charge, chromatin unwinds.
 acetyl group is removed from lysine by histone deacetylase enzymes (HDAC).

Question 7

2. Methylation of histones.

Answer 7

 methylation can also target arginine (R) residues
“ does not reduce positive charge of aa
“ occur in all core histones (H2A, H2B, H3 & H4)
 catalysed by histone methylase enzymes

Question 8

3. Ubiquitination of histones.

Answer 8

 ubiquitin links only to histone H2A or H2B

 only at a single internal lysine in each histone, some distance away from Me and Ac groups

 ubiquitination reduces the positive charge of the histone

Question 9

4. Sumoylation of histones.

Answer 9

 sumoylation leads to recruitment of HDAC enzymes & hence deacetylation of histone

Question 10

5. Phosphorylation of histones.

Answer 10

 phosphate molecule is negatively charged, ie will partially neutralize positive charge of histones

Question 11

Structural and functional domains in chromatin.

Functional domain: Locus-control regions (LCR)

Answer 11

 regulate the chromatin structure of a large region of DNA

 are a tissue-specific control elements on DNA

 ensures optimal expression of genes that fall under its control

 main mode of action is by altering chromatin structure

 ensures that all the genes under its control are in an open conformation and ready for transcription

Question 12

Functional domain: Locus-control regions (LCR).

Example: organisation of the beta-globin gene locus.

Answer 12

LCR region is 10 – 20 kb upstream of gene cluster.
• LCR activates individual genes in a tissue-specific manner during embryonic development
LCR can be linked to an individual beta-globin gene and expressed in transgenic mice, confers a high level of position-independent expression.
• inactivation of LCR in humans results in lack of expression of any of the genes in the cluster, even though the genes and their promoters or enhancers are intact

Question 13

LCR causes chromatin to adopt a more open configuration in specific cell types.

Answer 13

(a) if gene with an LCR is artificially inserted into genome, it will be organised into open chromatin relative to flanking DNA, can be transcribed
(b) inserted gene without LCR is influenced by adjacent regions and in a closed conformation; repressed

Question 14

How does LCR control expression of correct beta-globin gene during embryonic development?

Answer 14

• Individual beta-globin genes are expressed sequentially during erythroid development
• gene cluster organizes as an active chromatin hub
• at appropriate stage, looping of cluster brings together the regulatory elements of the particular gene to be transcribed and the regulatory elements in the LCR
• early: two gamma-globin genes are close to LCR  causes transcriptional activation
• later association involves sigma and beta-globin genes

Question 15

Insulators block the inappropriate spread of particular chromatin structures.

Answer 15

 insulators flank, for example an LCR and its associated genes. This ensures that only those genes are optimally expressed due to effect of LCR.
 regions outside are protected from its effect.

 loss of insulator causes spread of heterochromatin into adjacent areas, will then prevent expression of a gene

 heterochromatin: very tightly packed form of chromatin

 euchromatin: most of the DNA in the cell, contains DNA regions

Question 16

Eukaryotic gene regulation involves positive mechanisms.

Answer 16

 most genes are switched off (inactive) as a rule

 particular gene is selectively activated when needed

e.g. in different cells / stages of development / environments…